Taraxacum officinale as an expression system for recombinant proteins : molecular cloning and functional analysis of the genes encoding the major latex proteins [[Elektronische Ressource]] / vorgelegt von Florence Foucu

rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen - Florence B

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121 pages

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Biologie

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Publié par	rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
Publié le	01 janvier 2006
Nombre de lectures	25
Langue	English
Poids de l'ouvrage	8 Mo

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Taraxacum officinale as an expression system for recombinant
proteins:
Molecular cloning and functional analysis of the genes encoding the
major latex proteins

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von Master of Biology
Florence Foucu
aus Pau, Frankreich

Berichter: Universitätprofessor Rainer Fischer
Universitätprofessor Dirk Prüfer
Tag der mündlichen Prüfung: 27. November 2006

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

i

This thesis is dedicated to my family

iiACKNOWLEGDEMENT

First of all, I want to express my gratitude to Prof. Rainer Fischer and Prof. Dirk Prüfer for
giving me the opportunity to work on my PhD in this captivating subject.

I am grateful to my supervisor, Jost Muth, for guiding this work so well and for being so
helpful and supportive all along of this adventure.

I thank Thomas Rademacher who challenged my ideas and results, revised them, made them
more precise; for his helpful advices and discussions that allowed me to develop the
techniques necessary to carry this project though to fruition.

This work would not have been possible without the help of Flora Schuster to whom I am
grateful for the enormous patience and diligence in her care of my precious plants.

Many thanks to Elsa Arcalis and Julian Rodriguez for their precious help and brilliant
expertise in microscopy.

I thank Raphael Soeur and Michael Küpper for their expertise in protein analysis and mass
spectrometry.

In preparing this thesis, I owe much to my colleagues, especially Nicole Raven and Louisa
Bortesi for their helpful suggestions and criticisms. I would like to give a special
acknowledgment to Jost and Duncan for critically reviewing this thesis.

And least but not last, I want to give a huge thank you to Caroline, Vicky, Jutta, Nicole,
Louisa, Thomas, Julian, Jörg, and all the people from the Fraunhofer IME and the Institute of
Biologie VII for their helpful discussions and making life easier and funnier.

I thank Prof. Dirk Prüfer and Prof. Fritz Keuzaler for agreeing to act as co-examiners.

iiiTABLE OF CONTENT
I INTRODUCTION ______________________________________________ 1
I.1 Definition of molecular farming ___________________________________________ 1
I.2 Advantage of plants in molecular farming ___________________________________ 1
I.3 Existing plant systems ___________________________________________________ 3
I.4 Rationale for producing foreign proteins in the latex ___________________________ 4
I.5 Taraxacum officinale latex as a carrier for recombinant proteins __________________ 6
I.6 Reporter genes to monitor the regulation of MLPs expression ____________________ 8
I.7 Expression of complex heterologous proteins_________________________________ 9
I.8 Objective and strategy of the study ________________________________________ 11
I.8.1 Objective of the study ________________________________________________ 11
I.8.2 Experimental design _________________________________________________ 11
II MATERIAL AND METHODS ____________________________________ 13
II.1 Material ___________________________________________________________ 13
II.1.1 Organisms13
II.1.1.1 Plant material_______________________________________________________ 13
II.1.1.2 Bacterial strains_____________________________________________________ 13
II.1.2 Nucleic acids _________________________________________________________ 13
II.1.2.1 Vectors13
II.1.2.2 Primers14
II.1.3 Antibodies___________________________________________________________15
II.1.4 Chemicals and consumables _____________________________________________ 15
II.1.5 Stock solutions and buffers ______________________________________________ 16
II.1.6 Media and additives____________________________________________________ 17
II.1.6.1 Plant media ________________________________________________________ 17
II.1.6.2 Media for bacterial cultivation _________________________________________ 18
II.1.6.3 Antibiotics_________________________________________________________19
II.1.6.4 X-Gal and IPTG_____________________________________________________ 19
II.1.6.5 Substrate stock solution for the enzymatic assays and GUS reporter protein______ 19
II.1.7 Enzymes and reaction kits _______________________________________________ 19
II.1.8 Matrices and membranes ________________________________________________ 20
II.1.9 Equipment and applications____________________________________________ 20
II.2 Methods ___________________________________________________________ 22
II.2.1 Protein analysis _______________________________________________________ 22
ivII.2.1.1 Plant protein extraction _______________________________________________ 22
II.2.1.2 SDS-PAGE ________________________________________________________ 22
II.2.1.3 Protein quantification ________________________________________________ 22
II.2.1.4 Edman degradation __________________________________________________ 23
II.2.1.5 Protein sequence analysis _____________________________________________ 23
II.2.1.6 Plant specimen processing for microscopy staining and PPO activity assays _____ 23
II.2.1.7 Westernblot 24
II.2.1.8 Dot blot analysis ____________________________________________________ 24
II.2.1.9 Surface plasmon resonance (SRP) analysis________________________________ 24
II.2.1.10 Mass spectrometry___________________________________________________ 24
II.2.2 Nucleic acid technologies _______________________________________________ 25
II.2.2.1 Isolation of genomic DNA from T. officinale ______________________________ 25
II.2.2.2 DNA quantification__________________________________________________ 25
II.2.2.3 Agarose gel electrophoresis of DNA_____________________________________ 25
II.2.2.4 Isolation of ppo gene fragments by inverse PCR ___________________________ 25
II.2.2.5 RT-PCR of ppo cDNA from the latex____________________________________ 26
II.2.2.6 Rapid Amplification of the cDNA Ends (RACE) 26
II.2.2.7 Isolation of genomic flanking regions 27
II.2.2.8 PCR amplification of gene of interest 27
II.2.2.9 DNA sequence analysis_______________________________________________ 27
II.2.2.10 Restriction digest of DNA_____________________________________________ 28
II.2.2.11 Dephosphorylation__________________________________________________28
II.2.2.12 Ligation of DNA ____________________________________________________ 28
II.2.2.13 Isolation of plasmid DNA from E.coli ___________________________________ 28
II.2.2.14 Preparative agarose gel electrophoresis __________________________________ 28
II.2.3 Bacterial genetic manipulation ___________________________________________ 29
II.2.3.1 Preparation of electrocompetent E. coli cells ______________________________ 29
II.2.3.2 Transformation of E. coli by electroporation 29
II.2.3.3 Culturing of E. coli and glycerol stock preparation _________________________ 29
II.2.3.4 Expression and purification of the PPO carboxyl domain ____________________ 29
II.2.3.5 Polyclonal antibody production_________________________________________ 30
II.2.4 Plant genetic manipulation ______________________________________________ 30
II.2.4.1 Transformation of Agrobacterium by electroporation _______________________ 30
II.2.4.2 Transient gene expression _____________________________________________ 30
II.2.4.3 Stable gene expression in T. officinale plants and growing conditions___________ 31
II.2.5 Reporter gene assays ___________________________________________________ 31
II.2.5.1 DsRed fluorescence__________________________________________________ 31
vII.2.5.2 Confocal microscopy_________________________________________________ 32
II.2.5.3 Histochemical staining for GUS activity__________________________________ 32
II.2.5.4 Fluorometric assays for GUS detection___________________________________ 32
II.2.6 Immunolocalisation studies ______________________________________________ 33
II.2.6.1 Specimen processing for immuno-fluoresecence and electron microscopy _______ 33
II.2.6.2 Immuno-fluorescence microscopy ______________________________________ 33
II.2.6.3 Immunogold labeling________________________________________________33
III RESULTS___________________________________________________ 34
III.1 Identification and characterisation of the Major Latex Proteins __________________ 34
III.1.1 Identification of the Major Latex Proteins___________________________________ 34
III.1.2 Development and distribution of the laticifers in T. officinale ___________________ 36
III.1.3 Polyphenol oxidase activity assays ________________________________________ 37
III.2 Molecular cloning and expression analysis of the lat